ALTERNATIVE SPLICE VARIANT PATTERNS OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE (hTERT) IN THYROID TUMORS TO DISTINGUISH BENIGN FROM MALIGNANT

ABSTRACT

This invention relates, e.g., to a method for determining if a thyroid tumor in a subject is malignant, comprising determining in a sample from the subject the amount of TERT (telomerase reverse transcriptase) mRNA which lacks the β sequence and the amount of TERT mRNA in the sample which comprises the β sequence, wherein a preponderance (e.g., at least about 55%) of TERT mRNA in the sample which comprises the β sequence indicates that the tumor is malignant, and wherein a preponderance of TERT mRNA which lacks the β sequence indicates that the tumor is not malignant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/746,418 filed Jun. 4, 2010, now U.S. Pat. No. 9,222,136 granted onDec. 29, 2015, which is a 35 U.S.C. §371 U.S. national entry ofInternational Application PCT/US2008/013456, having an internationalfiling date of Dec. 5, 2008, which claims the benefit of U.S.Provisional Application No. 61/005,593, filed Dec. 5, 2007, the contentof each of the aforementioned applications is herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.CA095703, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND INFORMATION

The clinical problem associated with patients who present with asuspicious thyroid nodule continues to place clinicians and patients insituations where decisions about the surgical approach need to be madewith inadequate information. Although fine needle aspiration (FNA)biopsy of a thyroid nodule is very sensitive in the detection ofmalignancy, it is indeterminate or suspicious in 20-30% of cases. Thereare over 100,000 patients each year who present with a suspiciousthyroid nodule in the United States. Terminologies commonly used insuspicious cytopathology reports include the following: follicular orHürthle cell neoplasm, suspicious for papillary or follicular variant ofpapillary thyroid cancer, or cellular atypia. Because clinicians oftencannot determine malignancy, either pre- or intra-operatively, patientswith suspicious thyroid lesions cannot be optimally managed. This oftenresults in two scenarios: 1) patients who ultimately have a benignlesion on final histopathology may be subjected to unnecessary surgery;2) patients with a malignant thyroid nodule may need to undergo a secondoperation for completion thyroidectomy only after a diagnosis of canceris rendered on permanent histological section. Thus, there is a need fora diagnostic test that can distinguish more effectively betweenmalignant and non-malignant thyroid tumors, and that can provideguidance as to whether aggressive treatment, such as a totalthyroidectomy, should be administered.

Telomerase is a ribonucleoprotein complex that stabilizes linearchromosomes (e.g. human chromosomes) by adding telomere sequence(TTAGGG) repeats to their ends. The protein component of this complex,the telomerase reverse transcriptase catalytic subunit (TERT), has beencharacterized in a variety of species. The human form of the protein isdesignated as hTERT (human telomerase reverse transcriptase). The wildtype hTERT mRNA contains 16 exons. In addition, alternative splicing ofRNA transcribed from the hTERT DNA has been observed. Seven alternativesplice sites have been reported for hTERT, giving rise to splicevariants that may include three deletions and four insertions. See,e.g., JP Venables (2004) Cancer Res 64, 7647-7654; Kilian et al, (1997)Hum Mol Genet 6, 2011-2019; Killin et al., U.S. Pat. No. 6,916,642). Thesplicing patterns are presented schematically in FIGS. 1A and 1B herein.There are several possible combinations of these alternative splicesites resulting in a large number of potential variant transcripts, butonly a few have been confirmed (Hisatomi et al. (2003) Neoplasia 5,193-197). The sequence of the wild type hTERT mRNA, as used herein, isrepresented by SEQ ID NO:1 (taken from U.S. Pat. No. 6,916,642).Variants of this sequence, including updated sequences, polymorphisms,allelic variants, or the like, are also included. The numbering of thesequence of SEQ ID NO:1 is used herein to indicate the location of thesplice sites. The sequence of the polypeptide translated from SEQ IDNO:1 is represented by SEQ ID NO:2.

The four insertions and one deletion (deletion, 182 nt) generated by thealternative splices result in premature termination and non-functionalproteins (Hisatomi et al. (2003) (supra)). The β-deletion, in whichexons 7 and 8 are deleted, at nucleotides (nt) 2286-2468, gives rise toa reading frame-shift at nucleotide 2287, which is joined to nucleotide2469, and a subsequent termination codon at nucleotide 2605. The hTERTprotein translated from this alternatively spliced mRNA is thustruncated. The 182 nt deleted β sequence (sometimes referred to hereinas the β-deletion) is represented by SEQ ID NO:3; the protein sequencetranslated from it is inactive and is represented by SEQ ID NO:4. Thetranslation product of an mRNA having the α-splice (36 bp deleted withinthe RT motif A, extending from nt 2131-2166) has been shown in cellculture studies to be a dominant negative inhibitor of telomeraseactivity (Wick et al. (1999) Gene 232, 97-106). The sequence of thisα-deletion (sometimes referred to herein as the α-sequence) isrepresented by SEQ ID NO:5; the polypeptide translated from it isrepresented by SEQ ID NO:6. The γ-deletion (189 bp) has been identifiedin hepatocellular carcinoma cell lines and is also believed to benon-functional (Kilian et al, (1997) (supra)).

Telomerase enzyme activity has been reported by several groups to beregulated by posttranscriptional alternative splicing of hTERT (See,e.g., Colgin et al. (2000) Neoplasia 2, 426-432; Fan et al. (2005) ClinCancer Res 11, 4331-4337). Furthermore, the patterns of hTERTalternative splice variants are known to vary in ovary, kidney, uterineand breast cancer, compared to corresponding adjacent normal tissues(See, e.g., Colgin et al. (2000) (supra); Ulaner et al. (1998) CancerRes 58, 4168-4172; Ulaner et al. (2000) Int J Cancer 85, 330-335;Yokoyama et al. (2001) Mol Hum Reprod 7, 853-857). To our knowledge, nostudies have reported differences between alternative splice variantpatterns in benign and malignant tumors that originate from the sametissue type, or splice variant patterns that are more specific markersof malignant or benign disease than overall hTERT transcript levels.Comparable TERT alternative splicing patterns, including the α and the βdeletions, have been characterized from vertebrate species other thanhuman; the precise locations of the splice sites and the sequences ofthe wild type transcript are readily available to a skilled worker.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B shows a diagram of hTERT alternative splice variants. FIG. 1Ashows the structure of hTERT deletion variants. Locations oftelomerase-specific T motif, 7 conserved reverse transcriptase (RT)motifs (1, 2, A, B′, C, D, and E), exons 3-13, and deletion sites areindicated. In FIG. 1B, the alternative splice sites (α-,β-,γ-) aredepicted with the respective resulting transcripts. Primers F1720 andR3071 were used for the first PCR reaction. Nested primers F2162 andR2580 were used to amplify the region containing the α- and β deletions,resulting in four possible PCR products. Nested primers F2653 and R2932were used to amplify the region containing the γ-deletion, resulting twopossible PCR products.

FIG. 2A-2B shows hTERT alternative splice variant patterns in thyroidtumors. FIG. 2A shows gels demonstrating hTERT alternative splicevariant patterns present in representative thyroid tumors. The one blanklane in FA represents an hTERT negative tumor. FIG. 2B shows plots ofrelative proportions of full-length (F), α-deletion and β-deletiontranscripts for the 8 thyroid tumor types (only tumor types thatexhibited the combination of α-β-deletion are labeled β-/α-/β-). AN,adenomatoid nodule; FA, follicular adenoma; FC, follicular carcinoma;FVPTC, follicular variant of papillary thyroid carcinoma; HA, Hüthlecell adenoma; HC, Hüthle cell carcinoma; LcT, lymphocytic thyroiditisnodule; PTC, papillary thyroid carcinoma.

FIG. 3A-3B shows telomerase enzyme activity in a subset of 16 thyroidtumors. FIG. 3A shows telomerase activity found in benign vs. malignanttumors. FIG. 3B shows telomerase activity found in tumors with differenthTERT splice patterns, full-length, α-, β-/α-β- or hTERT negative.Results are plotted in arbitrary units. The long horizontal barsindicate means; vertical bars, the standard error of the means; and theshort horizontal bars, standard deviations. (ν), malignant tumors; (O),benign tumors.

FIG. 4 shows c-Myc gene expression vs. hTERT alternative splice variantpatterns. The ratios of c-myc and GAPDH mRNA levels determined by realtime RT-PCR are shown for the three categories of hTERT splice variants.Long horizontal bars represent means; vertical bars, the standard errorof the means; and short bars, standard deviations. (ν), malignanttumors; (O), benign tumors.

FIG. 5A shows the splice array probe configuration for each known andpredicted splicing event. FIG. 5B shows the configuration for intronretention splicing event (as prepared by Exonhit Therapeutics, Inc.).

DESCRIPTION OF THE INVENTION

The present inventors show herein that an hTERT mRNA alternative splicevariant in which the 182 nucleotide (nt) sequence from nt 2286-nt 2468(the β sequence) has been deleted is disproportionately present inthyroid tumors that are non-malignant, whereas the presence of the βsequence in an hTERT mRNA is characteristic of thyroid tumors that aremalignant. This 182 bp sequence (sometimes referred to herein as theβ-deletion or the β-sequence) is represented as SEQ ID NO:3. [As usedherein, the term “a TERT mRNA” (e.g., an hTERT mRNA) refers to an mRNAthat has been transcribed from a TERT gene (e.g., an hTERT gene). A TERTmRNA can be one of the wild type spliced mRNAs, or it can be analternatively spliced variant (sometimes referred to herein as an ASV).]This observation by the inventors provides the basis for an assay todetermine if a thyroid tumor in a subject is malignant; the differenttypes of RNAs (or proteins encoded by them) can serve as diagnosticmarkers for whether a thyroid tumor is malignant or benign. An assay ofthe invention can be used, e.g., to classify a thyroid tumor as beingmalignant or benign, to monitor the response to a treatment of a thyroidtumor, to identify an agent for treating a thyroid tumor, or otherapplications which will be evident to a skilled worker.

Additional spliced mRNAs from other genes, whose presence or absence isdiagnostic of malignant thyroid tumors, are also disclosed.

Advantages of a method of the invention include that it is rapid,inexpensive, and accurate. The differential diagnosis of a thyroid tumorcan, e.g., prevent a subject having a benign tumor from having toundergo unnecessary surgery, and can allow for a subject found to have amalignant tumor to undergo only a single operation (a totalthyroidectomy) to remove the entire thyroid. This is particularly truein the case in which prior screening by standard cytological analysis ofa fine needle aspirate (FNA) has classified the tumor as being“indeterminate,” “suspicious,” or “inadequate.

One aspect of the invention is a method for determining if a thyroidtumor in a subject is malignant, comprising determining in a sample fromthe subject the amount of TERT (telomerase reverse transcriptase) mRNAwhich lacks the β sequence and the amount of TERT mRNA in the samplewhich comprises the β sequence, wherein a preponderance (e.g., at leastabout 55%) of TERT mRNA in the sample which comprises the β sequenceindicates that the tumor is malignant (likely to be malignant), andwhereas a preponderance (e.g., at least about 55%) of hTERT mRNA whichlacks the β sequence indicates that the tumor is not malignant (likelynot to be malignant).

In one embodiment of this method, the subject is human; the TERT mRNA ishTERT mRNA; and the β sequence is the 182 bp sequence represented by SEQID NO:3.

In one embodiment, a ratio of at least about 0.55 (e.g., at least about0.59) of the amount of hTERT mRNA which contains the sequence of SEQ IDNO:3 compared to the total amount of hTERT mRNA which either contains orwhich lacks the sequence of SEQ ID NO:3 indicates that the tumor ismalignant; and a ratio of at least about 0.55 (e.g., at least about0.59) of the amount of hTERT mRNA which lacks the sequence of SEQ IDNO:3 compared to the total amount of hTERT mRNA which contains or whichlacks the sequence of SEQ ID NO:3 indicates that the tumor is notmalignant.

In another embodiment, the method further comprises determining theamount of mRNA which lacks the α sequence (represented by SEQ ID NO:5)and/or the amount of mRNA which lacks both the α sequence and the βsequence are determined; wherein,

a ratio of the amount of hTERT mRNA which contains the sequence of SEQID NO:3 compared to the total amount of

-   -   hTERT mRNA which contains the sequence of SEQ ID NO:3, and    -   hTERT mRNA which lacks the sequence of SEQ ID NO:3, and    -   hTERT mRNA which lacks the α sequence (SEQ ID NO:5) and/or which        lacks both SEQ ID NO:3 and SEQ ID NO:5)

of at least about 0.55 (e.g., at least about 0.59) indicates that thetumor is malignant, and

a ratio of the amount of hTERT mRNA which lacks the sequence of SEQ IDNO:3 compared to the total amount of

-   -   hTERT mRNA which contains the sequence of SEQ ID NO:3, and    -   hTERT mRNA which lacks the sequence of SEQ ID NO:3, and    -   hTERT mRNA which lacks the α sequence (SEQ ID NO:5) and/or which        lacks both SEQ ID NO:3 and SEQ ID NO:5

of at least about 0.55 (e.g., at least about 0.59) indicates that thetumor is not malignant.

In one aspect of the invention, the amount of each of the TERT mRNAs isdetermined by a method comprising amplifying mRNA in the sample byreverse transcriptase polymerase chain reaction (RT-PCR), using suitablePCR primers to amplify each mRNA species of interest; and detecting theamounts of the amplified products. The amounts of the amplified productscan be measured by a method comprising (a) subjecting the amplifiedproducts to a sizing procedure and categorizing the amplified productson the basis of their size; and/or (b) hybridizing the amplifiedproducts to suitable nucleic acid probes which are specific for theβ-sequence or for a control sequence that is present in TERT mRNAs whicheither comprise, or lack, the β-sequence.

In another aspect of the invention, the amount of each of the TERT mRNAsis determined by a method comprising quantitative real time PCR.

In another aspect of the invention, wherein the sample is a tissuesample or a fine needle aspirate (FNA), wherein the amount of each ofthe hTERT mRNAs is determined by a method that comprises performing insitu hybridization of the sample with suitable probes that are specificfor the β-sequence, or that are specific for a control sequence that ispresent in TERT mRNAs which either comprise, or lack, the β-sequence.

In another aspect of the invention, the amount of each of the hTERTmRNAs is determined by a method that comprises measuring the amounts ofpolypeptides translated from each of the mRNAs. For example, thepolypeptides can be measured by reacting them with antibodies that arespecific for epitopes within the β-sequence or that are specific forcontrol epitopes that are present in polypeptides translated from TERTmRNAs which either comprise, or lack, the β-sequence.

A method as above can further comprise: (a) analyzing the sample for thepresence of a BRAF mutation, wherein the presence of the mutation isfurther indicative that the tumor is malignant; and/or (b) determiningthe level of expression in the sample of one or more of the genes HMGA2,PLAG1, CDH3, SPOCK1, CEACAM6, DPP4, PRSS3, PDE5A, LRRK2, RAG2, AGTR1 orTP05, compared to the level in a benign tumor, wherein a statisticallysignificant amount of over-expression of one of more of genes HMGA2,PLAG1, CDH3, SPOCK1, CEACAM6, DPP4, PRSS3, PDE5A or LRRK2 furtherindicates that the tumor is malignant, and a statistically significantamount of under-expression of one or more of RAG2, AGTR1 or TP05 furtherindicates that the tumor is not malignant; and/or (c) determining thelevel of the spliced RNA species listed in Tables 3 and 4, compared tothe level in a benign tumor, wherein a significantly increased amount ofone or more of the spliced species in Table 3 further indicates that thetumor is malignant, or a significantly increased amount of one of moreof the spliced species in Table 4 indicates that the tumor is benign.The level of expression of the proteins can be determined by measuringthe amount of mRNA transcribed from the genes, or the amount of proteintranslated from the mRNA.

A method as above can further comprise, if the tumor is determined to bemalignant, performing a total thyroidectomy on the subject, or, if thetumor is determined not to be malignant, not performing a totalthyroidectomy on the subject. A method of the invention can be a methodfor deciding on a treatment modality: if a tumor is determined to bemalignant, a decision is made to perform a total thyroidectomy on thesubject, but if a tumor is determined not to be malignant, a decision ismade not to perform a total thyroidectomy on the subject.

One aspect of the invention is a method for treating a subject having athyroid tumor, comprising determining by a method of the inventionwhether the tumor is malignant and,

if the tumor is malignant, treating the subject aggressively for thyroidcancer, and

if the tumor is determined not to be malignant, not treating the subjectaggressively for thyroid cancer.

In one aspect of the invention, the method is carried out both before orat approximately the same time as, and after, the administration of atreatment for thyroid cancer, and is a method for determining theeffectiveness of the treatment.

This invention relates, e.g., to a method for determining if a thyroidtumor in a subject is malignant, comprising measuring in a sample fromthe subject the amounts, compared to a baseline value, or compared toeach other, of wild type transcripts and/or splice variant transcriptsof the telomerase reverse transcriptase (TERT) gene and/or one or moreof the of the genes listed in Tables 3 and 4, wherein the amount of thetranscript(s) compared to the baseline value (or compared to each other)indicates whether the tumor is malignant or benign. The baseline valuecan be any value that reflects the difference between the expression ofthe transcript(s) in a malignant tumor compared to a non-malignant(benign) tumor. The TERT gene can be from any vertebrate, including ahuman. Although much of the discussion herein is directed to humansubjects (e.g., patients) and human telomerase reverse transcriptase(hTERT), it will be evident to a skilled worker that non-human subjects,and other forms of TERT, are also included.

By a “sample” (e.g. a test sample) from a subject having a thyroid tumoris meant a sample that is suspected of comprising malignant thyroidtumor cells. The sample may be, e.g., from a biopsy of a thyroid tumor(e.g., a fine needle aspirate, or FNA). Furthermore, it is expectedthat, like most cancers, tumor cells from the thyroid are shed into theblood stream. Therefore, blood samples (e.g., plasma or serum) can beassayed by a method of the invention. Lymph node samples (e.g., FNAs)can also be assayed.

Methods for obtaining samples and preparing them for analysis (e.g., fordetection of the amount of an mRNA or of a protein translated from themRNA) are conventional and well-known in the art.

A “subject,” as used herein, includes any vertebrate that has a thyroidtumor. Suitable subjects (patients) include laboratory animals (e.g.,mouse, rat, rabbit, monkey, or guinea pig), farm animals (e.g., cattle,horses, pigs, sheep, goats, etc.), and domestic animals or pets (e.g.,cats or dogs). Non-human primates and, preferably, humans, are included.

One embodiment of the invention is a method for determining if a thyroidtumor in a subject (e.g., a human subject) is malignant, comprisingmeasuring in a sample from the subject the amount of TERT (e.g., for ahuman subject, hTERT) mRNA which lacks the β-sequence, and the amount ofTERT mRNA which contains the β-sequence, and determining from therelative amounts of the mRNA lacking or having the β-sequence whetherthe tumor is malignant. A preponderance (e.g., at least about 55%, or atleast about 59%) of TERT mRNA in the sample that comprises theβ-sequence indicates that the tumor is malignant, whereas apreponderance (e.g., at least about 55%, or at least about 59%) of TERTmRNA in the sample which lacks the β-sequence indicates that the tumoris not malignant (is benign).

“About,” as used herein, refers to plus or minus 10%. Thus, “about” 55%includes 49.5%-60.5%, so a lower limit of “at least about 55%” includesat least 49.5%; and “about” 59% includes 53.1%-64.9%, so a lower limitof “at least about 59%” includes at least 53%. “About” also refers toplus of minus 10% when referring to lengths of polynucleotides orpolypeptides. When a value is non-divisible, such as the number ofnucleotides or amino acids, and the value is not an integer, it will beevident to a skilled worker that the nearest integer is meant.

Because assays in the biomedical field are rarely 100% accurate, as usedherein an assay that indicates that a tumor is malignant indicates thatthe tumor is likely to be malignant. That is, the tumor has at leastabout a 70% chance (e.g., at least about an 80% or a 90% chance) ofbeing malignant. For example, as is shown in the Examples, a ratiogreater than about 0.55 (e.g., greater than about 0.59) of hTERT mRNAhaving the β-sequence, compared to the total amount of hTERT mRNA(having or not having this sequence) provides a specificity of 90%,indicating that the presence of such a ratio suggests that a tumor hasat least about a 90% chance of being malignant.

In one embodiment of the invention, the amount of TERT mRNA (e.g., inthe case of humans, hTERT mRNA) which lacks the β-sequence (e.g., inhumans, SEQ ID NO:3) is compared to the sum of hTERT transcripts in thesample which do and do not include the β-sequence, wherein apreponderance (e.g., at least about 55%) of TERT transcripts whichcontain the β-sequence indicates that the tumor is malignant, whereas apreponderance (e.g., at least about 55%%) of TERT transcripts which lackthe β-sequence indicates that the tumor is not malignant.

In another embodiment, the amount of an mRNA or interest (eithercomprising or lacking the β-sequence) is compared to the amounts of oneor more of the following types of mRNA molecules: mRNAs having theα-deletion, and/or having the β-deletion, and/or having both theα-deletion and the β-deletion, and/or having neither of these deletions.For example, the amount of an mRNA of interest can be compared to thetotal amount of all four of these types of mRNA.

Instead of, or in addition to, comparing a TERT mRNA of interest to thetotal amounts of TERT mRNA within a given sample which comprise, orlack, the β-sequence, one can compare the amount of the mRNA of interestto the amount of a control mRNA within the sample. For example, one cannormalize the amount of a TERT mRNA of interest to a constitutivelyproduced mRNA, such as actin, tubulin, or the like. Consider now ahypothetical example, in which the amount of β-spliced TERT mRNA in atest sample, as normalized to such an internal control, is compared tothe amount of β-spliced TERT mRNA, normalized to a comparable control,from a pool of thyroid tumors or cells in culture which are known to bebenign or known to be malignant. The values from the pool of tumors orcells may be available in a database compiled from the values, and/orthey may be determined based on published data or on retrospectivestudies of patients' tissues, and other information as would be apparentto a person of ordinary skill implementing a method of the invention.Because it can be difficult to use actual patient samples in a clinicalenvironment, reference standards, such as RNA (or DNA) produced in vitro(e.g., recombinantly), or defined amounts of a purified or semi-purifiedRNA (or DNA) can be used. The normalized amount of β-spliced TERT mRNArepresenting the level in a benign tumor, or the normalized amount ofβ-spliced TERT mRNA representing the level in a in a malignant tumor,can serve as a baseline value. Upper and lower baseline values(reference standards) can be used. Baseline values may be selected usingstatistical tools that provide an appropriate confidence interval sothat measured levels that fall outside the standard value can beaccepted as being aberrant from a diagnostic perspective, and predictiveof the presence (or absence) of malignancy.

In the hypothetical example above, consider the case in which the amountof β-spliced mRNA in the test sample is statistically the same (orhigher) than the baseline value corresponding to benign thyroid tumors.This indicates that the tumor is likely to be benign. However, if theamount of the β-spliced mRNA in the test sample is statisticallysignificantly lower than this baseline value, this indicates that thetest tumor is likely to be malignant. Alternatively, a baseline valuecan be determined on the basis of a subject, population of subjects,etc., which are known to have malignant thyroid tumors. In this case, ifthe amount of β-spliced mRNA in a test sample is statistically the same(or lower) than the baseline value corresponding to malignant thyroidtumors, then the test tumor is likely to be malignant. However, if theamount of the β-spliced mRNA in the test sample is statisticallysignificantly higher than the baseline value, the test tumor is likelyto be benign.

A “significant” increase or decrease in the amount of an mRNA orprotein, as used herein, can refer to a difference which is reproducibleor statistically significant, as determined using statistical methodsthat are appropriate and well-known in the art, generally with aprobability value of less than five percent chance of the change beingdue to random variation. Some such statistical tests will be evident toa skilled worker, and some are discussed in the Example herein. Forexample, a significant increase in the amount of mRNA or proteincompared to a baseline value can be at least about 50% higher (e.g., atleast about 2-fold, 5-fold, 10-fold, or more higher).

In one embodiment of the invention, the thyroid tumor being tested issuspected of being malignant. For example, the thyroid tumor can havebeen classified as being suspicious (for malignancy) or as beingindeterminate, based on a cytological assay, such as a cytological assayperformed on a sample obtained from a fine needle aspirate (FNA). Fordiscussions of what criteria are used to categorize a thyroid tumor assuspicious or indeterminate, and the methods for carrying out a FNAcytological assay, see, e.g., Banks et al. (2008) Thyroid 18, 933-941;Baloch et al. (2002) Diag Cytopathol 26, 41-44; or Yoder et al. (2006)Thyroid 16, 781-786.

A variety of methods can be employed to determine the amounts of theTERT mRNA species in a sample.

In one embodiment of the invention, the amount of an mRNA of a giventype (such as a particular splice variant of interest) is measureddirectly, without further amplification. For example, the presence of asplice or the length of an mRNA can be determined by Northern analysis,a probe protection assay, mass spectroscopy, or other conventionalmethods. Appropriate probes for such methods will be evident to askilled worker. For example, for an RNAse probe protection assay todistinguish a wild type hTERT mRNA from a TERT mRNA having a particularsplice variant, the probe can be a DNA fragment having sequencescorresponding to the junction of the wild type (non-deleted) sequenceand the alternative intron/exon sequence or derived from the sequencesurrounding the alternative intron/exon deletion site. For example, aDNA fragment consisting of sequences of the wild type hTERT mRNA thatspan nucleotides 2286-2287 (e.g., a fragment consisting of nucleotides2236-2336) will protect the wild type mRNA sequence as a 101 ntfragment, but will protect an RNA with the β-splice as a 51 nt fragment.Fragments for RNAse probe protection are usually chosen in the range of30 to 400 bases and are positioned to yield readily distinguishableprotection products.

In another embodiment of the invention, the amounts of the mRNAs aredetermined indirectly, by a method comprising reverse transcribing theminto cDNAs; amplifying the cDNAs by any of a variety of suitablemethods, using suitable primers; and detecting the amounts of theamplified product(s). Among the well-known amplification methods thatcan be used are, e.g., the polymerase chain reaction (PCR) which, whencarried out in conjunction with the reverse transcriptase step issometimes referred to as RT-PCR, quantitative or semi-quantitative realtime PCR, ligase chain reaction DNA signal amplification, amplifiableRNA reporters, Q-beta replication, transcription-based amplification,boomerang DNA amplification, strand displacement activation, cyclingprobe technology, isothermal nucleic acid sequence based amplification,or other self-sustained sequence replication assays.

For amplification assays, primer pairs can be used that either flank thealternative intron/exons or require the presence of the alternativeintron/exon for amplification. Suitable primers can be designed based onthe sequences presented herein, in view of the known splice sitepositions. Generally, the primer pairs are designed to generate anamplification product of an easily detectable size. The primers may onlyallow amplification of a single alternative intron/exon. For example, atleast one primer of a primer pair may be specific for a sequence withinthe 182 nt β-sequence. If the second primer of the primer pair is alsospecific for a sequence in the β-sequence, then only TERT mRNAs thatcomprise this sequence will be amplified. Similarly, if the secondprimer lies 5′ or 3′ to the first primer, only TERT mRNAs that comprisethe β-sequence will be amplified. In another embodiment, two primersflanking the β-sequence can be used, and the size of the resultingamplification product will indicate if the β-sequence is present orabsent. In another embodiment, at least one primer of a primer pair isspecific for a sequence that spans the intron/exon junction of analternative splice site (e.g., that spans nucleotides 2286-2287, such asa primer comprising nucleotides 2276-2296). In this case, only RNAs thatare not spliced at this site will be amplified.

In some circumstances, detection of multiple alternative intron/exons,and/or wild type intron/exons, in the same RNA preparation, may becarried out. For example, it may be useful to amplify a sequence thatcontains both the β-sequence (if present) and a nearby sequence that ispresent in hTERT mRNAs that either comprise the β-sequence or lack thissequence. Nearby sequences can be amplified, e.g., by a forward primerthat lies 5′ to the 5′ end of the β-sequence, and/or a reverse primerthat binds to a sequence in exon 9 or 10. Amplification of the nearbysequences can be used to determine if an mRNA lacking the β-sequence ispresent in the sample being tested. Alternatively, separate, controlprimer pairs can be used to amplify either the β-sequence (if present)or a control sequence that is present in TERT mRNAs that either do or donot comprise the β-sequence. In some embodiments, a longer TERT mRNA isfirst amplified, and then nested primers are used to amplify sequenceswithin the first amplification product. A typical such set of nestedamplifications is described in the Examples herein. Other suitablecombinations of broader plus nested amplification reactions will beevident to a skilled worker.

Suitable amplification primers (e.g., pairs of PCR primers) can bedesigned by conventional methods. If desired, conventional softwareprograms can be employed to aid in designing the primers.Oligonucleotides used as amplification primers (e.g., DNA, RNA, PNA,LNA, or the like) preferably do not have self-complementary sequences orhave complementary sequences at their 3′ end (to prevent primer-dimerformation). Preferably, the primers have a GC content of about 50% andmay contain restriction sites to facilitate cloning. Amplificationprimers can be between about 10 and about 100 nt in length. They aregenerally at least about 15 nt and not longer than 50 nt, although insome circumstances and conditions shorter or longer lengths can be used.For example, primers from between about 15 and about 35 nucleotides canbe used. Amplification primers can be purchased commercially from avariety of sources, or can be chemically synthesized, using conventionalprocedures. Some exemplary PCR primers that can be used to detectspliced variants of hTERT are described, e.g., in the Examples herein,as well as in Stein Saeboe-Larssen et al. (2006) BMC Molecular Biology7, 26; Kilian et al, (1997) Hum Mol Genet 6, 2011-2019; and Killin etal., U.S. Pat. No. 6,916,642.

PCR primers are annealed to cDNA and sufficient amplification cycles,generally about 20-40 cycles, are performed to yield a product that isreadily detected, e.g. by gel electrophoresis and staining. Methods ofPCR amplification, and reagents used therein, are conventional. Forguidance, see, e.g., PCR Protocols: A Guide to Methods and Applications(Innis et al. eds, Academic Press Inc. San Diego, Calif. (1990)). Theseand other molecular biology methods used in methods of the invention arewell-known in the art and are described, e.g., in Sambrook et al.,Molecular Cloning: A Laboratory Manual, current edition, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & sons, New York, N.Y.

If desired, a detectable label, such as a radiolabel, biotinylatedlabel, fluorphor, chemiluminescent label, or the like, may be includedin an amplification reaction. Suitable labels include fluorochromes,e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red,phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); radioactivelabels, e.g., ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

Another method for detecting mRNAs utilizes quantitative (orsemi-quantitative) real-time PCR, using, for example, molecular beaconsor FRET (fluorescence resonance energy transfer). The FRET techniqueutilizes molecules having a combination of fluorescent labels which,when in proximity to one another, allows for the transfer of energybetween labels. See, e.g., the Examples herein or “iQ5 Real Time PCRDetection System” Manual (Bio-Rad, Hercules, Calif.).

The presence and amounts of the individual amplification products can bedetermined by a variety of procedures, including sizing them (e.g., bygel electrophoresis, capillary electrophoresis, Southern blot analysis,sequencing, high performance liquid chromatography, mass spectroscopy,etc.)

Alternatively, or in conjunction with a sizing procedure, the amplifiedDNA products can be hybridized to suitable detectable nucleic acidprobes, which are specific for one or more sequences that are present(or absent) in an mRNA of interest.

Probes for hybridization are generally at least about 15, 20, or 25nucleotides, but may range from about 10 to a full-length sequence. Theprobes may comprise additional sequences that do not hybridize to a DNAor an mRNA (or portion thereof) of interest. Probes are generally DNA,but may be RNA, PNA, LNA or derivatives thereof. Hybridization probesmay be labeled with a radiolabel, chemiluminescent label, or any of themyriad other known labels, such as those discussed above in relation toamplification primers. Electrochemiluminescence or laser-inducedfluorescence may be used.

For example, to detect the presence or absence of the β-sequence, theamplified products can be hybridized to a probe comprising at leastabout 10 (e.g., at least 15, 20, 25, 30, 35, 40 or as many as all)contiguous nucleotides of the β sequence (SEQ ID NO:3), or to completecomplements thereof, under conditions in which the hybridization isspecific. As technology improves, it may be possible to utilize probesthat are even shorter than 10 nts. If desired, a control probe can beused which is specific for a sequence that is present in all TERTtranscripts, such as a sequence from the exon 4, 5, 9 or 10 region. Forexample, an amplified DNA product can be hybridized to a sequencespecific for the β region and to a control sequence from elsewherewithin the TERT transcript. A DNA to which the TERT control as well asthe β probe hybridize reflects a TERT RNA that comprises the β sequence,whereas a DNA to which the TERT control but not the β probe hybridizes,reflects a TERT RNA that lacks the β sequence. Other suitable internalhybridization controls will be evident to a skilled worker.

Probes and conditions are selected, using routine conventionalprocedures, to insure that hybridization of a probe to a sequence ofinterest is specific. Methods for designing nucleic acid probes that arespecific for a nucleic acid of interest are conventional and well knownin the art. The TERT nucleic acid sequences disclosed herein, incombination with the splice maps, can be used to design probes that arespecific for any splice variant of interest.

A probe that is “specific for” a nucleic acid (e.g., an mRNA or a cDNA)contains sequences that are substantially similar to (e.g., hybridizeunder conditions of high stringency to) one of the strands of thenucleic acid. By hybridizing “specifically” is meant herein that the twocomponents (the mRNA or cDNA and the nucleic acid probe) bindselectively to each other and not generally to other componentsunintended for binding to the subject components. The parametersrequired to achieve specific binding can be determined routinely, usingconventional methods in the art. Probes that bind specifically to atarget of interest do not necessarily have to be completelycomplementary to them. For example, a probe can be at least about 95%identical to the target, provided that the probe binds specifically tothe target under defined hybridization conditions, such a conditions ofhigh stringency.

As used herein, “conditions of high stringency” or “high stringenthybridization conditions” means any conditions in which hybridizationwill occur when there is at least about 95%, preferably about 97 to100%, nucleotide complementarity (identity) between a nucleic acid ofinterest and a probe. Generally, high stringency conditions are selectedto be about 5° C. to 20° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH.Appropriate high stringent hybridization conditions include, e.g.,hybridization in a buffer such as, for example, 6× SSPE-T (0.9 M NaCl,60 mM NaH₂PO₄, 6 mM EDTA and 0.05% Triton X-100) for between about 10minutes and about at least 3 hours (in one embodiment, at least about 15minutes) at a temperature ranging from about 4° C. to about 37° C.). Inone embodiment, hybridization under high stringent conditions is carriedout in 5×SSC, 50% deionized Formamide, 0.1% SDS at 42° C. overnight.

Hybridization can be performed on preparations that are affixed to asolid support or in solution, to in situ tissue preparations, etc. Onetype of hybridization analysis is annealing to oligonucleotide probeswhich are immobilized on a suitable surface, such as a functionalizedglass slide, a nylon support, or a chip, e.g. in an array. Hybridizationconditions are chosen that are appropriate for the length andcomposition of the probe and the method of hybridization.

Other conventional methods to detect (e.g., quantify) amplified nucleicacids will be evident to a skilled worker. These include, e.g., ELISAdetection using biotinylated or modified primers, dot blotting,differential hybridization, subtractive hybridization, or the like.

In another embodiment of the invention, the amounts of the mRNAs aredetermined indirectly by measuring the amount of polypeptide translatedfrom the mRNAs. Generally, in such methods, antibodies are used whichare specific for a region of interest in the polypeptide.

As noted above, alternative intron/exon a, located from nucleotides2131-2166 can be spliced out of hTERT mRNA. A polypeptide translatedfrom such an RNA is deleted for 12 amino acids (which are represented bySEQ ID NO:6); this deletion removes reverse transcriptase motif A. Thepresence or absence of this spliced mRNA can be determined, e.g., byreacting polypeptide in a sample from a subject with an antibody that isspecific for an epitope within this 12 amino acid sequence, underconditions in which the antibody reacts specifically with polypeptidesthat comprise this epitope. Another of the variant sequences, theβ-deletion at nts 2286-2468, encodes a truncated protein, due to areading frame-shift at base 2287, which is joined to nt 2469, andsubsequently a termination codon at nt 2605. This variant protein hasreverse transcriptase domains 1, 2, A, B′, and part of C. In order todetect the presence or absence of this deletion, one can reactpolypeptides in a sample from a subject with an antibody that isspecific for an epitope within the 61 amino acid polypeptide translatedfrom the deleted sequence (this 61 amino acid sequence is represented bySEQ ID NO:4). Alternatively, one can use an antibody specific for aminoacids that lie downstream of the termination codon; mRNAs having theβ-deletion will not generate a polypeptide having this sequence, whereasmRNAs having the β-sequence will encode and translate those amino acidsequences. In any of these assays, it is preferable to reactpolypeptides in the sample from the subject with a positive controlantibody, which will hybridize to a portion of the TERT protein that isexpected to be present in proteins translated from both RNAs which lackand which comprise the deleted sequence. For example, antibodies can beused which are specific for epitopes of polypeptides translated fromexon 4 (RT domains 1 and 2). Other suitable positive control antibodieswill be evident to a skilled worker.

Antibodies suitable for use in assays of the invention are commerciallyavailable, or can be prepared routinely. Methods for preparing and usingantibodies in assays for polypeptides of interest are conventional, andare described, e.g., in Green et al., Production of Polyclonal Antisera,in Immunochemical Protocols (Manson, ed.), (Humana Press 1992); Coliganet al., in Current Protocols in Immunology, Sec. 2.4.1 (1992); Kohler &Milstein (1975), Nature 256, 495; Coligan et al., sections 2.5.1-2.6.7;and Harlow et al., Antibodies: A Laboratory Manual, page 726 (ColdSpring Harbor Laboratory Pub. 1988).

Any of a variety of antibodies can be used in methods of the invention.Suitable antibodies include, e.g., polyclonal, monoclonal (mAbs),recombinant, humanized or partially humanized, single chain, Fab, andfragments thereof. The antibodies can be of any isotype, e.g., IgM,various IgG isotypes such as IgG_(1′) IgG_(2a), etc., and they can befrom any animal species that produces antibodies, including goat,rabbit, mouse, chicken or the like. The term, an antibody “specific for”a polypeptide, means that the antibody recognizes a defined sequence ofamino acids, or epitope, in the polypeptide, and binds selectively tothe polypeptide and not generally to polypeptides unintended for bindingto the antibody. The parameters required to achieve specific binding canbe determined routinely, using conventional methods in the art.Antibodies are generally accepted as specific against telomerase proteinif they bind with a K_(d) of greater than or equal to 10⁻⁷ M, preferablygreater than of equal to 10⁻⁸ M. The affinity of a monoclonal antibodyor binding partner can be readily determined by one of ordinary skill inthe art (see, e.g., Scatchard (1949) Ann. N.Y. Acad. Sci. 51, 660-6672).

In one embodiment of the invention, antibodies specific for a (one ormore) polypeptide of the invention are immobilized on a surface (e.g.,are reactive elements on an array, such as a microarray, or are onanother surface, such as used for surface plasmon resonance (SPR)-basedtechnology, such as Biacore), and polypeptide or regions of interest ina polypeptide in the sample are detected by virtue of their ability tobind specifically to the antibodies. Alternatively, polypeptides in thesample can be immobilized on a surface, and detected by virtue of theirability to bind specifically to the antibodies. Methods of preparing thesurfaces and performing the analyses, including conditions effective forspecific binding, are conventional and well known in the art. In oneembodiment of the invention, the antibody is contacted with ahistological preparation (e.g. from a thyroid tumor or lymph nodebiopsy), and the amount of polypeptide is determined byimmunohistochemical staining (e.g., in situ).

Among the many types of suitable immunoassays are immunohistochemicalstaining, immunocytochemical staining, ELISA, ELISPOT, Western blot(immunoblot), immunoprecipitation, radioimmuno assay (RIA),immunofluorescence (e.g., fluorescence-activated cell sorting (FACS)),immunoprecipitation, etc. Assays used in a method of the invention canbe based on colorimetric readouts, fluorescent readouts, massspectroscopy, visual inspection, etc. Assays can be carried out, e.g.,with suspension beads, or with arrays, in which antibodies or cell orblood samples are attached to a surface such as a glass slide or a chip.

A method of the invention can be combined with additional tests todetermine if a thyroid tumor is malignant. For example, a sample can befurther tested to determine if it contains a mutation in the BRAF gene(a serine-threonine kinase), wherein the presence of the mutation isfurther indicative that the tumor is malignant. (See, e.g., Cheng et al.(1998) Br J Cancer 77, 2177-2180.) Alternatively, a method of theinvention can be combined with an assay for any of the splice variantsshown in Tables 3 and 4, wherein a significantly increased amount of oneor more of the splice variants in Table 3, or a significantly decreasedamount of one or more of the splice variants in Table 4 furtherindicates that the thyroid tumor is malignant. Moreover, a sample can befurther tested to determine if it contains a significantly increased ordecreased amount of expression of one of the genes that the presentinventors have shown to be correlated with malignancy of thyroid tumors.In a paper recently published by some of the present inventors and theircolleagues (Prasad et al. (2008) Clin Cancer Res 14, 3327-37), ninegenes were identified which are statistically over-expressed inmalignant thyroid tumors (HMGA2, PLAG1, CDH3, SPOCK1, CEACAM6, DPP4,PRSS3, PDE5A and LRRK2), and three genes were identified which arestatistically under-expressed in malignant thyroid tumors (RAG2, AGTR1and TP05). The degree of expression of these genes can also be used tofurther determine whether a thyroid tumor being tested is malignant orbenign.

In one embodiment of the invention, if a subject is determined by amethod of the invention to be likely to have a malignant thyroid tumor,a decision can be made to treat the subject with an aggressive form oftreatment; and, in one embodiment, the aggressive treatment is thenadministered. Suitable aggressive treatment modalities include, forexample, a total or near-total thyroidectomy and optionally, followingthe surgery, treatment with radioactive iodine or treatment with atargeted agent. By contrast, if a subject is determined not to be likelyto have a metastatic tumor, a decision can be made not to treat thesubject further, or to adopt a less aggressive treatment regimen. In oneembodiment, the subject is then treated with less aggressive forms oftreatment. Suitable less aggressive forms of treatment, which areappropriate for benign lesions, include, for example, a thyroidlobectomy. A subject that does not have a metastatic thyroid tumor isthus spared the unpleasant side effects associated with the unnecessary,more aggressive forms of treatment. By “treated” is meant that aneffective amount of an agent such as radioiodine or other anti-cancerprocedure is administered to the subject. An “effective” treatmentrefers to a treatment that elicits a detectable response (e.g. atherapeutic response) in the subject.

A detection (diagnostic) method of the invention can be adapted for manyuses. For example, it can be used to monitor the response to atreatment. For example, after a total thyroidectomy has been performedand the subject has been treated with radioactive iodine or anothertreatment, a sample from the subject (e.g., blood or a lymph nodesample) can be assayed to determine the relative amount of TERT mRNAwhich comprises or lacks the β sequence. A subject can be monitored inthis way to determine the effectiveness for that subject of a particulardrug regimen; or a drug or other treatment modality can be evaluated ina pre-clinical or clinical trial. In these methods, a relative increasein the amount of TERT mRNA lacking the β sequence compared to the amountof TERT mRNA comprising the sequence is indicative of effectivetreatment.

A method of the invention can be adapted to identify an agent fortreating a thyroid tumor. In one embodiment, a population of thyroidcells (e.g., cells in culture or in a tumor in an animal model, such asa conventional mouse model for thyroid cancer) that has been determinedby a method of the invention to be malignant is contacted with a testagent; and the mRNA expression pattern is determined after a designatedperiod of time of treatment with the agent. An agent that can alter theexpression pattern to be more like the expression pattern of anon-malignant thyroid tumor is a candidate for an agent to treatmalignant thyroid cancer.

One aspect of the invention is a kit for detecting whether a thyroidtumor is likely to be malignant, comprising one or more agents fordetecting the amount of a spliced mRNA of the invention (e.g., the bymeasuring the amount of the mRNA, and/or the amount of a polypeptideencoded by it). As used herein, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.For example, “an” mRNA of the invention, as used above, includes 1, 2,3, 4, 5 or more of the mRNAs. The agents in the kit can encompass, e.g.,probes specific for the mRNA that can be used to hybridize to the RNA(or to a cDNA or PCR product generated from it) or specific primers forperforming RT-PCR, or antibodies specific sequences of interest in thepolypeptides. The kit may also include additional agents suitable fordetecting, measuring and/or quantitating the amount of nucleic acid orpolypeptide. Among other uses, kits of the invention can be used inexperimental applications. A skilled worker will recognize components ofkits suitable for carrying out a method of the invention.

Optionally, a kit of the invention may comprise instructions forperforming the method. Optional elements of a kit of the inventioninclude suitable buffers, containers, or packaging materials. Thereagents of the kit may be in containers in which the reagents arestable, e.g., in lyophilized form or stabilized liquids. The reagentsmay also be in single use form, e.g., for the performance of an assayfor a single subject.

In the foregoing and in the following examples, all temperatures are setforth in uncorrected degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

EXAMPLES Example I Materials and Methods A. Tumor Tissues

One hundred and thirty three thyroid tumors were collected under JohnsHopkins Institutional Review Board approval from patients undergoingthyroid surgery. Samples included 60 malignant (28 papillary thyroidcancers, 24 follicular variant of papillary thyroid cancers, 5follicular cancers, and 3 Hürthle cell cancers) and 73 benign lesions(31 adenomatoid nodules, 21 follicular adenomas, 12 Hürthle celladenomas, and 9 Hashimoto's thyroiditis nodules). Follicular and Hürthlecell cancers are relatively infrequent thyroid tumors resulting in thelimited sample numbers. Samples were snap frozen in liquid nitrogen andstored at −80° C. until use. Among these 133 samples, a subset of 50tumors had suspicious FNA cytology reports.

B. RT-and Nested PCR

Total RNA was isolated from each tumor with Trizol (Invitrogen,Carlsbad, Calif.) and purified with RNeasy Mini Kit (Qiagen, Valencia,Calif.). Reverse transcription was performed with 1 μg of total RNA andoligo(dT) primers by SuperScript II reverse transcriptase (Invitrogen).hTERT alternative splice variants were amplified by nested PCR usingprimers designed according to GenBank accession No. AF015950 (FIG. 1B).The first round of amplification spanned a region that included all α-,β-, and γ-deletion sites with forward primer F1720,5′-GCTGCTCAGGTCTTTCTTTTAT-3′ (SEQ ID NO:7) and reverse primer R3071,5′-GGAGGATCTTGTAGATGTTGGT-3′ (SEQ ID NO:8). PCR was performed in 25 μlof reaction mixture using 1 μl of the cDNA, Platinum Taq DNA polymerase(Invitrogen) by incubation at 94° C. for 2 minutes, followed by 25amplification cycles of 94° C. for 30 seconds, 54° C. for 30 seconds,and 72° C. for 90 seconds, and a final extension at 72° C. for 5minutes. The second round of PCR was carried out with 1 μl of the firstround PCR product, nested primer sets, and the Platinum Taq DNApolymerase. The nested primer set for hTERT α- and β-transcriptvariants, forward F2162 5′-CCGCCTGAGCTGTACTTTGTC-3′ (SEQ ID NO:9) andreverse R2580 5′-CAGAGCAGCGTGGAGAGGAT-3′ (SEQ ID NO:10), produced fourpossible products: α⁺β⁺ (418-bp), α⁻β⁺(382-bp), α⁺β⁻ (236-bp), and α⁻β⁻(200-bp) respectively by incubation at 94° C. for 2 minutes, followed by25 amplification cycles of 94° C. for 20 seconds, 57.3° C. for 20seconds, and 72° C. for 30 seconds, and a final extension at 72° C. for2 minutes. The hTERT γ-transcript was amplified using the nested primerset, forward F2653 5′-GGTGGATGATTTCTTGTTGGT-3′ (SEQ ID NO:11) andreverse R2932 5′-GGTGAGACTGGCTCTGATGG-3′ (SEQ ID NO:12), yielding 2possible products: 280- (γ⁺) and 91-bp (γ) in length, by incubation in asimilar fashion with the exception of a different annealing temperatureof 55.5° C. Amplified products were electrophoresed on 2% agarose gelswith Nucleic Acid Gel Stain (Cambrex, Rockland, Me.) and visualizedunder ultraviolet light. The densitomeiric value of each hTERTtranscript was quantified using Quantity One image analysis software(version 4.5.2; BioRad, Hercules, Calif.). The relative gene expressionlevel of each transcript was reported as a relative proportion of allthe hTERT transcripts present in the same sample (28). GAPDH served asan internal control.

C. Statistical Analysis

For analysis of the hTERT alternative splice variant data in thyroidtumors, the following comparisons were performed: 1) between malignant(n=60) and benign (n=73) thyroid tumors and, 2) between malignant (n=19)and benign (n=31) thyroid lesions that had corresponding suspicious orindeterminate FNA cytology. These cytologies included: suspicious forpapillary thyroid cancer or follicular variant of papillary thyroidcancer, thyroid neoplasm, follicular neoplasm, Hürthle cell neoplasm,and neoplasm. Because the data were recorded as the proportion oftranscripts in each respective gel lane (full-length, α-, andβ-/α-β-deletion), a comparison of equal proportions between tumor typeswas done. This comparison was based on a standardized differencestatistic in multinomial probabilities and tested using a permutationapproach. For the purpose of analysis, the α-β-deletion was consideredin the same category as the β-deletion since both variants producenon-functional proteins.

D. Receiver Operating Characteristic (ROC) Analysis

An ROC analysis was done to evaluate the use of relative proportions ofhTERT splice variants to classify tumors as either benign or malignant.The following three splice variants were quantified: 1) full-lengthhTERT transcript; 2) α-deletion transcript and 3) β-/α-β-deletiontranscript (β-/α-β-deletion was defined as the sum of relativeproportions for β- and α-β-deletion transcripts). Since the three ROCcurves corresponding to each transcript (full, α- and β-/α-β-deletions)were from the same sample, the method of Delong et al [(1988) Biometrics44, 837-845] was implemented for the comparison of estimated areas undereach curve. Once a transcript variant was identified as a preferablediagnostic tool, thresholds were reported for 1) simultaneouslymaximizing sensitivity and specificity (Gallop et al. (2003)Understanding Statistics 2, 219-242) and, 2) maximizing specificitywhile also retaining a sensitivity greater than 50%. This secondapproach was chosen to minimize the probability of false positives,since FNA already provides a high level of sensitivity.

E. Quantitative Telomerase Enzyme Activity Assay

Telomerase enzyme activity assay was performed on a subset of 16 of the133 samples using the Quantitative Telomerase Detection Kit (US Biomax,Inc, Ijamsville, Md.) and according to the manufacturer's instructions.Briefly, for each sample, protein from twelve 10 μm cryosections wasextracted in 100 μl CHAPS lysis buffer at 4° C. The proteinconcentration was determined using Bio-Rad Protein Assay (Bio-RadLaboratories). Heat-inactivated controls were performed bypre-incubating extracts at 85° C. for 10 minutes. For each assay 1 μgprotein was added to a 25 μl QTD reaction mix. Reactions were performedin 96-well plates on an ABI prism 770-sequence detector. The extensionreactions were run for 20 minutes at 25° C., followed by 40 cycles ofPCR amplification and a melting curve analysis performed. A standardcurve was constructed using a dilution series of the telomerase standardsubstrate provided by the manufacturer and used to calculate relativeamounts of the TRAP assay product. The reaction products were thenelectrophoresed on a 10% polyacrylamide gel and the telomerase hexamerladders visualized by ethidium bromide staining.

F. Real-Time PCR for c-myc

Real-time RT-PCR for c-myc was performed on a subset of 23 of the 133samples using the synthesized first-strand cDNA from total RNA isolatedfrom thyroid tumors. Assays-on-demand Gene Expression products were usedfor c-myc (Hs00153408_ml) and GAPDH (Hs99999905_ml) (Applied Biosystems,Foster City, Calif.). Reactions were performed in a 20 μl reactionvolume containing 1× Taq Man universal PCR master mix (AppliedBiosystems), 1× Gene expression assay mix (primers and TaqMan MGB probedye-labeled with FAM) and 1 μl cDNA. Reactions were performed on anABI7300HT sequence detection system machine (Applied Biosystems). AllPCR reactions were performed in triplicate. Fluorescence was quantifiedwith the Sequence detection system software, version 2.0 (AppliedBiosystems).

Example II Results of Studies of hTERT Alternative Splice Patterns

A. hTERT Alternative Splice Variant Patterns in Thyroid Tumors

hTERT gene expression was detected in 114 of the 133 (86%) thyroidtumors (Table 1).

TABLE 1 Thyroid Tumors Analyzed for hTERT alternative splice variantpatterns by subtype Malignant (n = 60) Benign (n = 73) PTC FVPTC FC HCFA AN HA LcT Final Histology (n = 28) (n = 24) (n = 5) (n = 3) (n = 21)(n = 31) (n = 12) (n = 9) hTERT positive 28 21 3 1 21 25 6 9 (n = 114)hTERT full-length > 0.33^(a) 24(86%) 4(19%) 3(100%) 1(100%) 1(5%) 1(4%)4(67%) 3(33%) (n = 41) ^(a)An hTERT full-length expression cut point of0.33 corresponded to a specificity of 0.85 and a sensitivity of 0.60.AN, adenomatoid nodule; FA, follicular adenoma; FC, follicularcarcinoma; FVPTC, follicular variant of papillary thyroid carcinoma; HA,Hürthle cell adenoma; HC, Hürthle cell carcinoma; LcT, lymphocyticthyroiditis nodule; PTC, papillary thyroid carcinoma

No tumor exhibited a γ-deletion splice variant and only 4/133 exhibitedan α-β-deletion variant. Representative gels are shown in FIG. 2A. ThehTERT splice variant patterns present in papillary thyroid cancers,follicular variant of papillary thyroid cancers, follicular adenomas,and adenomatoid nodules (the 4 most common tumor types that can besuspicious on thyroid FNA) are depicted. The gels demonstrate theprominent presence of full-length hTERT gene expression in papillarythyroid cancer and the progressive loss thereof in follicular variant ofpapillary thyroid cancer and the benign tumors (follicular adenoma andadenomatoid nodule). Note that “full-length” hTERT gene expression, asused in this Example, refers to mRNA corresponding to the PCR productgenerated by the nested primers, wherein the mRNA does not exhibit theα-, β-, or α plus β-deletions. There is also a concomitant gain of theinhibitory α-deletion, non-functional β- and α-β-deletion patterns inthe benign tumors.

B. Statistical Analysis of All Thyroid Nodules

Overall, we found significant differences in the proportions of thevarious transcripts between malignant and benign thyroid tumors(p<0.001). On average, the malignant tumors exhibited larger proportionsof full-length hTERT transcripts (0.57±0.15) than either the α-(0.13±0.02), or β-/α-β-deletion transcripts (0.30±0.11, FIG. 2B). Thiswas true for all malignant tumor types except follicular variant ofpapillary thyroid cancer. In contrast, the benign tumors exhibitedgreater proportions of β-/α-β-deletion transcripts (0.64±0.08) thaneither the full-length (0.19±0.06) or α-deletion transcripts (0.17±0.02,FIG. 2B).

C. Analysis of Suspicious Thyroid Nodules

In a subset analysis, we repeated our hTERT splice variant assay on 50thyroid tumors with the preoperative diagnosis of suspicious FNA (Table2), the cytological category most in need of additional moleculardiagnostic tools. Thirty-eight of the 50 (76%) were hTERT positive. Theresults in this subset were similar to the original cohort, withmalignant tumors exhibiting greater proportions of full-lengthtranscripts compared to α- and β-/α-β-deletion transcripts, while amongthe benign tumors (with the exception of Hürthle cell adenomas), greaterproportions of β-/α-β-deletion transcripts were observed compared tofull-length or α-deletion transcripts.

TABLE 2 hTERT Gene Expression in the Subset of Thyroid Nodules withPreoperative Suspicious FNA Diagnosis Malignant (n = 19) Benign (n = 31)PTC FVPTC FC HC FA AN HA LcT Final Histology (n = 4) (n = 9) (n = 5) (n= 1) (n = 14) (n = 4) (n = 8) (n = 1) hTERT positive (n = 38) 4 9 3 1 144 2 1 hTERT full-length > 0.59^(a) 4 1 3 1  1 0 1 0 (n = 11) ^(a)AnhTERT full-length expression cut point of 0.59 corresponded to aspecificity of 0.90 and a sensitivity of 0.53. AN, adenomatoid nodule;FA, follicular adenoma; FC, follicular carcinoma; FNA, fine needleaspiration; FVPTC, follicular variant of papillary thyroid carcinoma;HA, Hürthle cell adenoma; HC, Hürthle cell carcinoma; LcT, lymphocyticthyroiditis nodule; PTC, papillary thyroid carcinoma

D. Receiver Operating Characteristic (ROC) Analysis

Altogether, 114 cases that were hTERT gene expression-positive wereincluded in the ROC analysis. Since malignant tumors exhibited a greaterproportion of full-length transcripts, we focused on this transcript asa diagnostic tool, and this approach resulted in an area under the curve(AUC) of 0.79. Based on the simultaneous maximization method, afull-length transcript threshold of 0.22 corresponded to a sensitivityand specificity of 0.74. Similar results were observed for the 38 hTERTpositive samples from the subset with suspicious FNAs, with an estimatedAUC of 0.69 and, based on a full-length threshold of 0.17, a sensitivityand specificity of 0.67.

In addition to the above approach using equal maximization ofsensitivity and specificity, we also examined the full-length thresholdassociated with the largest observed specificity for a given sensitivityno less than 0.50. By applying these criteria to all samples, afull-length transcript threshold of 0.33 achieved a specificity of 0.85for a given sensitivity of 0.60 (Table 1). Among the subset ofsuspicious thyroid nodules, a full-length threshold of 0.59 correspondedto a specificity of 0.90 for a given sensitivity of 0.53 (Table 2),thereby providing a diagnostic strategy with a very high specificity.

E. Quantitative Telomerase Enzyme Activity Analysis

We also tested a subset of 16 thyroid tumors for functional telomeraseactivity. The malignant tumors (n=8) showed significantly higher averagetelomerase enzyme activity (FIG. 3A) than the benign samples (n=8) (ttest: p=0.03). Several of the thyroid cancers that exhibited minimalcapsular invasion or follicular variant of papillary thyroid cancermorphology had telomerase activity values similar to the benign samples.Furthermore, only alternative splice variant patterns showing apreponderance of full-length transcript were significantly associatedwith high levels of telomerase enzyme activity (χ² test: p=0.02, FIG.3B).

F. c-Myc Expression and hTERT Alternative Splice Variant Patterns

Next, we studied the correlations between c-myc and hTERT geneexpression. Similar to others, we observed a statistically significantassociation between c-myc and hTERT gene expression positive samples.However, we also documented that this correlation did not vary among thedifferent specific splice variant patterns (FIG. 4). Of the 23 samplestested for c-myc gene expression (13 malignant and 10 benign), thefollowing three groups were defined: 1) full-length hTERT (n=8); 2) α-and β-/α-β-deletion variants hTERT (n=9); and 3) negative hTERT (n=6). Acomparison of mean differences between each pair of groups, with respectto c-myc gene expression, was conducted based on a chi-squared test. Nodifference was seen between full-length and α- and β-/α-β-deletionvariants. However, a significant difference in mean c-myc was observedbetween negative and full-length hTERT groups (p=0.003) and betweennegative and α- and β-/α-β-deletion variant groups (p=0.018). These datasuggest that c-myc gene expression correlates with overall hTERT geneexpression, regardless of whether or not hTERT is expressed in itsactive form.

Discussion

We examined the patterns of hTERT alternative splice variants in aneffort to discern differences between benign and malignant thyroidtumors. Because hTERT expression was low in most of the samples, thetarget concentration produced from a single conventional PCR within 30cycles was often too low to be detected. Nonspecific products arefrequently generated by increasing the amplification cycles with asingle set of primers, even with a hot start. Furthermore, quantitativereal time PCR is not applicable for the evaluation of 4 different hTERTisoforms. We therefore chose to use nested PCR in order to: 1) increasethe sensitivity of the assay to be able to detect each splice variantand 2) as an effective solution to PCR nonspecificity and gene copylimitation. One major concern about nested PCR is that it does notmaintain a linear relationship between the amount of final amplifiedproduct and the amount of target sequence. Studies indicate, however,that nested PCR will retain its utility for quantitation if the firstround PCR is maintained in the exponential phase (Zieger et al. (2005) JSurg Oncol 89, 108-113). Indeed, quantitative nested real-time PCR assayhas been developed and used in some studies without apparent distortionin the amplified product ratio (Renshaw et al. (2002) Am J Clin Pathol117, 19-21). Furthermore, we also optimized our nested PCR reactionusing thyroid cell lines to ensure accurate product ratios. In ourstudy, primers specific for each of the hTERT isoforms were used in thenested PCR. Our results clearly demonstrate significant differences inthe patterns of functional and non-functional hTERT transcripts inbenign vs. malignant tumors.

With the exception of follicular variant of papillary thyroid cancer,the malignant tumors exhibited a greater proportion of the hTERTfull-length transcript compared to either the α-, or β-/α-β-deletions,whereas the benign tumors exhibited a greater proportion of theβ-/α-β-deletion transcripts compared to the full or α-deletion (FIG.2B). The fact that follicular variant of papillary thyroid cancer showedcomparably less full-length transcript than the other thyroidmalignancies is in keeping with the notion that the histologicalevaluation of these tumors is often problematic, with inter-observervariation present up to 60% of the time.

One objective for testing thyroid tumors for differences in hTERTpatterns was to improve the specificity of the clinically ambiguous FNAdiagnosis of suspicious thyroid lesions. In the 50 tumors that hadcorresponding suspicious FNA cytology, the same patterns seen in the 133tumors were observed with the exception of Hürthle cell adenomas.Indeed, ROC analysis revealed that a full-length transcript proportionover 0.33 yielded a specificity of 85% in the diagnosis of thyroidmalignancy. Furthermore, setting the cut point of the full-lengthtranscript proportion at 0.59 in the subset with suspicious FNA reportsyielded 90% specificity.

Example III Identification of Other Splice Variants Associated withMalignant or Non-Malignant Thyroid Tumors

-   A. Splice array analysis

Twenty one thyroid tumors were analyzed by splice array analysis. Threeeach of papillary thyroid cancer, follicular variant of papillarythyroid cancer, follicular cancer, adenomatoid nodule, follicularadenoma, Hürthle cell adenoma, and lymphocytic thyroiditis nodule, pluscorresponding normal thyroid samples were hybridized to Human GenomeWide SpliceArray™ (ExonHit Therapeutics, Inc., Gaithersburg, Md.) on theAffymetrix platform.

In brief, the splice array analysis was carried out as follows:

Transcript amplification and labeling: Amplified, labeled cDNA wasprepared using the NuGEN WT-Ovation™ Pico RNA Amplification System andthe FL-Ovation™ cDNA Biotin Module V2. First strand cDNA was preparedfrom total RNA using a unique first strand DNA/RNA chimeric primer mixand reverse transcriptase (RT). The primers have a DNA portion thathybridizes either to the 5′ portion of the poly (A) sequence or randomlyacross the transcript. RT extends the 3′ DNA end of each primergenerating first strand cDNA. Fragmentation of the mRNA within thecDNA/mRNA complex creates priming sites for DNA polymerase to synthesizea second strand, which includes DNA complementary to the 5′ uniquesequence from the first strand chimeric primers. The result is a doublestranded cDNA with a unique DNA/RNA heteroduplex at one end that isisothermally amplified using the SPIA™ process, developed by NuGEN™. Theprocess includes a SPIA™ DNA/RNA chimeric primer, DNA polymerase andRNase H in a homogeneous isothermal assay that provides highly efficientamplification of DNA sequences. An average mRNA amplification of15,000-fold is observed with 500 pg of starting total RNA.

Array hybridization, scanning, and data extraction: Standard methodsfollowing recommendations of the manufacturer were used to hybridize thesamples to the Splice Arrays. The arrays were stained and washed usingthe FS450-001 fluidics protocol prior to scanning with the AffymetrixGeneChip® Scanner 3000 7G. DAT and .CEL images were then visuallyinspected for anomalies and accurate grid placement.

Three samples from the malignant thyroid subtype, Hürthle cell cancer,did not pass quality control specifications and thus, were not tested.Briefly, total RNAs were isolated from tumor and normal thyroid and,reverse transcribed with random primers prior to PCR amplification. ThePCR products were then enzymatically fragmented and labeled at their 3′termini. The resulting products were hybridized to SpliceArrayscontaining over 6,000,000 probes representing 138,000 known or predictedsplice events (FIGS. 5A and 5B). The different splice events tested forinclude the following: alternative exons in which exons are either: 1)skipped or 2) included; 3) alternative 5′ (donor) and 4) 3′ (acceptor)splice sites; and; 5) retained introns.

-   B. Overall analytical approach: Given the complexity of analyses of    our exon expression data, the possibility of 5 different events for    each exon, and 7 different thyroid subtypes we chose the following    overall approach. We first selected probes with statistically    significant differential expression between each tumor type and    corresponding matched normal thyroid; these results were then used    to filter expression heterogeneity among subtypes within the same    class (malignant or benign) by applying a novel query-based    comparisons algorithm (Kowalski et al. From Ambiguities to Insights    in Cancer Diagnosis via Query-based Comparisons. Pattern    Recognition, 2008. doi:10.1016/j.patcog.2008.09.030). The objective    of this second screen was to select genes that characterized    specific tumor subtype pairs within each class to ultimately select    genes that characterize the benign and malignant classes overall.-   C. Normalization of Array Data: The array data were normalized by    Partek's Genomics Suite software while importing the 42 .CEL files.    Data were first processed using GC content background correction    followed by Robust Multichip Average (RMA) background correction    (Marme et al. (2008) Int J Cancer 123, 2048-56). Quantile    normalization was performed across all 42 arrays (21 tumors and 21    matched normal thyroid). Data were Log 2 transformed and mean probe    summarization was applied. The data set was filtered based on the    expression values' frequency distribution in order to remove probe    sets that were expressed at a low level. A probe was removed if all    of the samples' intensity values fell below the pre-determined Log 2    based value of (4.3).-   D. Within class and within subtype, probe analyses. Two-way Analysis    of Variance (ANOVA) models were used to perform statistical tests on    the filtered expression values, comparing each tumor type against    its respective matched normal tissue. The overall signal intensity    values showed a normal distribution following Partek default    processing for all samples analyzed. This comparison resulted in    lists of differentially expressed transcripts, based on a fold    change of 1.8 (p-value≦0.001). Within malignant tumor subtypes, 822    distinct Entrez gene IDs were selected as showing significantly    different splice variant expression in papillary thyroid cancers vs.    matched normal, 889 in follicular variant of papillary thyroid    cancer, and 885 in follicular cancer. For the benign tumor subtypes,    884 distinct gene IDs were selected as significantly different in    adenomatoid nodule tumors vs. matched normal; 550 in follicular    adenoma, 824 in Hürthle cell adenoma and 606 in lymphocytic    thyroiditis nodule.-   E. Within class and between-subtypes, heterogeneity analyses. Using    papillary thyroid cancer as the ‘common’ malignant thyroid tumor    subtype, we paired it with each other subtype (follicular variant of    papillary thyroid cancer and follicular cancer), in order to    identify significantly expressed splice variants that were common    between each pair. We then compared results across all pairs to    identify splice variants in common to all 3 malignant tumors. We    identified 69 distinct Entrez genes that were common to papillary    thyroid cancer and follicular cancer; 81 common to papillary thyroid    cancer and follicular variant of papillary thyroid cancer; and 25    common to all 3 subtypes (Table 3). Similarly, by using adenomatoid    nodule as the ‘common’ benign tumor subtype, we paired each other    subtype (follicular adenoma, Hürthle cell adenoma, and lymphocytic    thyroiditis nodule) and found 38 genes common to adenomatoid nodule    and follicular adenoma; 63 common to adenomatoid nodule and Hürthle    cell adenoma; 44 common to adenomatoid nodule and lymphocytic    thyroid nodule and 2 common to all benign subtypes (Table 4). The 25    genes that characterized malignant tumor subtypes were distinct from    the two selected as characterizing benign tumor subtypes. Within the    malignant samples, we performed PCA analyses among all splice    variants chosen after ANOVA analysis and, for comparison, among the    25 genes selected as characterizing the malignant class. The 25    genes selected accounted for 44% of variability in splice variant    expression among malignant samples, whereas all the spice variants    identified after the ANOVA analysis accounted for 30% of    variability.-   F. Biological function: Gene Ontology (GO) <http://geneontology.org>    analysis of the 25 genes associated with the malignant tumors and    the 2 associated with the benign tumors was performed using the    Spotfire platform (Tables 3 and 4). This GO analysis software    provides a p-value for whether or not the selected genes are    randomly represented for each of the many GO functional categories    compared to the 20,100 well-characterized genes on the arrays. The    25 malignancy genes represented the following functions (p<0.005):    positive regulation of kinase activity (n=3 genes) and receptor    activity (n=8); and cellular location categories (p=0.00184):    membrane (n=18), plasma membrane (n=7) and external side of plasma    membrane (n=3). The 2 benign genes represented the following    functions (p<0.005): extracellular matrix organization and    biogenesis (n=1), collagen fibril organization (n=1); extracellular    matrix structural constituent conferring tensile strength (n=1,    p=0.000125); and cellular location categories anchoring collagen    (n=1).-   G. Summary: In summary, based on 25 probes, we were able to capture    44% of variability in expression among samples from the 3 malignant    subtypes in comparison to 30% based on all probes. Of the 8 genes    known to be associated with different types of cancer (ADH1C, AOX1,    ETK, KIT, NRCAM, SYNE1. AKR1CL2, and RAINB1), 3 genes (KIT, NRCAM,    and SYNE1) have been reported to be significantly associated with    thyroid cancer; SYNE1 associated with epigenetic regulation; KIT, a    proto-oncogene that encodes a transmembrane receptor tyrosine    kinase; and NRCAM, a neuronal system cell-adhesion molecule.

TABLE 3 Exon events selected as commonly, differentially expressed amongmalignant thyroid tumor subtypes, PTC, FVPTC and FC vs. matched normaltissue. Entrez Gene Gene Chromosomal ID Symbol Location Event type 126ADH1C Chr 4q23 exon skipped 316 AOX1 Chr 2q33.1 alternative spliceacceptor, exon skipped 727 C5 Chr 9q33.2 exon skipped, intron 27 953ENTPD1 Chr 10q23.33 exon skipped, novel exons, alternative spliceacceptor, intron 4, intron 6 1804 DPP6 Chr 7q36.2 exon skipped, novelexon 2042 EPHA3 Chr 3p11.2-p11.1 exon skipped 3803 KIR2DL2 Chr 19q13.42alternative splice acceptor, alternative splice donor 3815 KIT Chr 4q12exon skipped, novel exon, intron 4 4897 NRCAM Chr 7q31.1 exons skipped,intron 5 6262 RYR2 Chr 1q43 exon skipped, intron 24, alternative spliceacceptor 8029 CUBN Chr 10p13 intron 55, alternative splice donor, intron13, intron 41, exon skipped 9162 DGKI Chr 7q33 intron 9, exon skipped,novel exon 9213 XPR1 Chr 1q25.3 exon skipped, intron 1, intron 14 9844ELMO1 Chr 7p14.2 exon skipped 9914 ATP2C2 Chr 16q24.1 exon skipped 10349ABCA10 Chr 17q24.3 exon skipped 23345 SYNE1 Chr 6q25.1-q25.2 intronretention, exon skipped, intron 62, intron 69 23348 DOCK9 Chr 13q32.3intron 46, exon skipped, novel exon, alternative splice acceptor 55755CDK5RAP2 Chr 9q33.2 intron 23, exon skipped, novel exon, intron 36 56899ANKS1B Chr 12q23.1 intron 12, exon skipped 83592 AKR1CL2 Chr 10p15.1exon skipped, intron 1, novel exons 89797 NAV2 Chr 11p15.1 novel exon,intron 24, exon skipped 93035 PKHD1L1 Chr 8q23.1-q23.2 exon skipped,intron 40, intron 41, intron 74, alternative splice donor 200879 LIPHChr 3q27.2 intron 7, exon skipped 223117 SEMA3D Chr 7q21.11 exonskipped, intron 12

TABLE 4 Exon events selected as commonly, differentially expressed amongbenign thyroid tumor subtypes, AN, FA, HA, and LcT vs. matched normaltissue. Entrez Gene Gene Chromosomal ID Symbol Location Event type 23223RRP12 Chr 10q24.1 exon skipped 1303 COL12A1 Chr 6q13-q14.1 exon skipped,intron 23, intron 27, intron 65

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions and to utilize the present invention to its fullest extent.The preceding preferred specific embodiments are to be construed asmerely illustrative, and not limiting of the scope of the invention inany way whatsoever. The entire disclosure of all applications, patents,and publications (including provisional patent application 61/005,593,filed Dec. 5, 2007) cited above and in the figures are herebyincorporated in their entirety by reference.

We claim:
 1. A method for determining if a thyroid tumor in a subject ismalignant, comprising determining in a sample from the subject theamount of TERT (telomerase reverse transcriptase) mRNA that lacks the βsequence and the amount of TERT mRNA in the sample that comprises the βsequence, wherein a preponderance of TERT mRNA in the sample thatcomprises the β sequence indicates that the tumor is malignant, andwherein a preponderance of TERT mRNA that lacks the β sequence indicatesthat the tumor is not malignant.
 2. The method of claim 1, wherein thesubject is human; the TERT mRNA is hTERT (human telomerase reversetranscriptase) mRNA; and the β sequence is the 182 bp sequencerepresented by SEQ ID NO:3.
 3. The method of claim 2, wherein a) a ratioof the amount of hTERT mRNA that comprises the sequence of SEQ ID NO:3,compared to the total amount of hTERT mRNA that either comprises or thatlacks the sequence of SEQ ID NO:3 of at least about 0.55 indicates thatthe tumor is malignant, and b) a ratio of the amount of hTERT mRNA thatlacks the sequence of SEQ ID NO:3, compared to the total amount of hTERTmRNA that either comprises or that lacks the sequence of SEQ ID NO:3 ofat least about 0.55 indicates that the tumor is not malignant.
 4. Themethod of claim 3, further wherein the amount of mRNA that lacks the αsequence (represented by SEQ ID NO:5) and/or the amount of mRNA thatlacks both the α sequence and the β sequence are determined; andwherein, a) a ratio of the amount of hTERT mRNA that comprises thesequence of SEQ ID NO:3 compared to the total amount of hTERT mRNA thatcomprises the sequence of SEQ ID NO:3, and hTERT mRNA that lacks thesequence of SEQ ID NO:3, and hTERT mRNA that lacks the α sequence (SEQID NO:5) and/or that lacks both SEQ ID NO:3 and SEQ ID NO:5 of at leastabout 0.55 indicates that the tumor is malignant, and b) a ratio of theamount of hTERT mRNA that lacks the sequence of SEQ ID NO:3 compared tothe total amount of hTERT mRNA that comprises the sequence of SEQ IDNO:3, and hTERT mRNA that lacks the sequence of SEQ ID NO:3, and 2hTERTmRNA that lacks the α sequence (SEQ ID NO:5) and/or which lacks both SEQID NO:3 and SEQ ID NO:5) of at least about 0.55 indicates that the tumoris not malignant.
 5. The method of claim 3, wherein the thyroid tumor issuspected of being malignant.
 6. The method of claim 5, wherein thethyroid tumor is classified as being suspicious for malignancy or asbeing indeterminate, based on a cytological assay.
 7. The method ofclaim 6, wherein the cytological assay is performed on a sample obtainedfrom a fine needle aspirate (FNA).
 8. The method of claim 3, wherein theamount of each of the hTERT mRNAs is determined by a method comprisingamplifying mRNA in the sample by reverse transcriptase polymerase chainreaction (RT-PCR) analysis, using suitable PCR primers to amplify eachof the mRNA species of interest; and measuring the amounts of theamplified products.
 9. The method of claim 8, wherein the amounts of theamplified products are measured by a method comprising a) subjecting theamplified products to a sizing procedure and categorizing the amplifiedproducts on the basis of their size; and/or b) hybridizing the amplifiedproducts to suitable nucleic acid probes which are specific for theβ-sequence or for a control sequence that is present in hTERT mRNAswhich either comprise, or lack, the β-sequence.
 10. The method of claim3, wherein the sample is a tissue sample or a fine needle aspirate(FNA), and wherein amount of each of the hTERT mRNAs is determined by amethod comprising performing in situ hybridization of the tissue sample,with suitable probes that are specific for the β-sequence or for acontrol sequence that is present in hTERT mRNAs which either comprise,or lack, the β-sequence.
 11. The method of claim 3, wherein the amountof each of the hTERT mRNAs is determined by a method comprisingmeasuring the amounts of polypeptides translated from each of the mRNAs.12. The method of claim 11, wherein the polypeptides are measured byreacting them with antibodies that are specific for epitopes within theβ-sequence or that are specific for control epitopes which are presentin polypeptides translated from hTERT mRNAs that either comprise, orlack, the β-sequence, under conditions which are effective for specificinteractions of the antibodies and their cognate epitopes.
 13. Themethod of claim 3, wherein the amount of each of the mRNA species isdetermined by quantitative real time PCR.